Biological Bypass Bridge with Sodium Channels, Calcium Channels and/or Potassium Channels to Compensate for Conduction Block in the Heart

ABSTRACT

This invention provides a bypass bridge comprising a tract of gap junction-coupled cells having a first end and a second end, both ends capable of being attached to two selected sites in a heart so as to allow the conduction of a pacemaker and/or electrical signal/current across the tract between the two sites, wherein the cells functionally express a sodium channel. The invention also provides related methods of making the bypass bridge, methods of implanting same in a heart, and methods of treating a disorder associated with an impaired conduction in a subject&#39;s heart.

This application claims the benefit of U.S. Provisional Application No.60/704,210, filed Jul. 29, 2005; 60/701,312, filed Jul. 21, 2005; and60/781,723 filed Mar. 14, 2006, the entire contents of which areincorporated herein by reference.

This invention was made with Government support under Grant No. HL-28958awarded by the National Institutes of Health. The Government has certainrights in the invention.

Throughout this application, various publications are referenced inparentheses by author name and date, patent number, patent applicationor publication number. Full citations for these publications may befound at the end of the specification immediately preceding the claims.The disclosures of these publications in their entireties are herebyincorporated by reference into this application in order to more fullydescribe the state of the art to those skilled therein as of the date ofthe invention described and claimed herein. However, the citation of areference herein should not be construed as an acknowledgement that suchreference is prior art to the present invention.

FIELD OF THE INVENTION

The present invention relates to a bypass bridge comprising a tract ofgap junction-coupled cells that functionally express a sodium channel,the two ends of the tract being attachable to two selected sites in aheart so as to allow the conduction of a pacemaker and/or pacemakerand/or electrical signal/current across the tract between the two sites.

BACKGROUND OF THE INVENTION

Electronic pacemakers are lifesaving devices that provide a regularheartbeat in settings where the sinoatrial node, atrioventricularconduction, or both, have failed. Thus, one of the major indications forelectronic pacemaker therapy is high degree heart block, such that anormally functioning sinus node impulse cannot propagate to theventricle. The result is ventricular arrest and/or fibrillation, anddeath.

Malfunction or loss of pacemaker cells can occur due to disease oraging. For example, acute myocardial infarction (MI) kills millions ofpeople each year and generally induces in survivors marked reductions inmyocyte number and cardiac pump function. Adult cardiac myocytes divideonly rarely, and the usual responses to myocyte cell loss includecompensatory hypertrophy and/or congestive heart failure, a disease witha significant annual mortality. There have been recent reports of thedelivery of bone marrow-derived and/or circulating human mesenchymalstem cells (hMSCs) to the hearts of post-myocardial infarct patientsresulting in some improvement of mechanical performance (Strauer et al.,2002; Perin et al., 2003) in the absence of overt toxicity. Thepresumption in these and other animal studies (Orlic et al., 2001) isthat the hMSC integrate into the cardiac syncytium and thendifferentiate into new heart cells restoring mechanical function.

An alternative application of cell therapy, described herein, involvesgrowing cells such as hMSCs into a bypass bridge comprising a tract ofgap junction-coupled cells that can be used to conduct pacemaker and/orelectrical current/signals across a region of the heart exhibitingimpaired electrical conduction.

SUMMARY OF THE INVENTION

The invention disclosed herein provides a bypass bridge comprising atract of gap junction-coupled cells having a first end and a second end,both ends capable of being attached to two selected sites in a heart soas to allow the conduction of a pacemaker and/or electricalsignal/current across the tract between the two sites, wherein the cellsfunctionally express a sodium channel. In certain embodiments of thebypass bridge, cells in the tract further functionally express apacemaker ion channel which induces a pacemaker current so as to inducea pacemaker current in the cells. In preferred embodiments, the firstend of the tract of cells is capable of being attached to the atrium andthe second end is capable of being attached to the ventricle, so as toform an atrioventricular (AV) bridge that allows conduction of apacemaker and/or electrical signal/current across the tract from theatrium to the ventricle.

The invention also provides a method of making a bypass bridge forimplantation in a heart comprising (a) transfecting a cell with, andfunctionally expressing therein, a nucleic acid encoding a sodiumchannel and optionally a nucleic acid encoding a pacemaker ion channel,and (b) growing the transfected cell into a tract of cells having afirst and a second end capable of being attached to two selected sitesin the heart, wherein the cells are physically interconnected viaelectrically conductive gap junctions.

The invention further provides a method of implanting a bypass bridge ina heart comprising (a) making a bypass bridge by any of the methodsdisclosed herein, (b) selecting a first and a second site in the heart,and (c) attaching the first end of the tract to the first site and thesecond end of the tract to the second site, so as to thereby implant abypass bridge in the heart that allows the conduction of a pacemakerand/or electrical signal/current across the tract between the two sites.

The present invention also provides a method of treating a disorderassociated with an impaired conduction in a subject's heart comprising(a) transfecting a cell with a nucleic acid encoding a sodium channel,wherein the cell functionally expresses the sodium channel, (b) growingthe transfected cell into a tract of cells having a first end and asecond end, wherein the cells are physically interconnected viaelectrically conductive gap junctions, (c) selecting a first site and asecond site in the heart between which sites conduction is impaired, and(d) attaching the first end of the tract to the first site and thesecond end of the tract to the second site, so as to allow theconduction of a pacemaker and/or electrical signal/current across thetract between the two sites and thereby treat the subject.

This invention further provides a method of treating a disorderassociated with an impaired conduction and impaired sinus node activityin a subject's heart comprising (a) transfecting a cell with at leastone nucleic acid encoding a sodium channel and a pacemaker ion channel,wherein the cell functionally expresses the sodium channel and thepacemaker ion channel, (b) growing the transfected cell into a tract ofcells having a first end and a second end, wherein the cells arephysically interconnected via electrically conductive gap junctions, (c)selecting a first site in the left atrium of the heart and a secondsite, between which sites conduction is impaired, and (d) attaching thefirst end of the tract to the first site and the second end of the tractto the second site, so as to allow the propagation of a pacemaker and/orelectrical signal/current generated by the sinus node and/or tract ofcells between the two sites and thereby treat the subject.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Alignment of Mouse and Human Hcn2 Polypeptide Sequences.

The mouse and human HCN2 polypeptide sequences are aligned for maximumcorrespondence. The amino acid sequences of mHCN2 and hHCN2 are setforth in SEQ ID NO: 14 and SEQ ID NO: 16, respectively.

FIG. 2. Amino Acid Sequence of the Human HCN212 Chimeric Channel.

The shaded N-terminal portion of the sequence is derived from hHCN2; theunderlined intramembranous portion from hHCN1; and the C-terminalportion (without shading or underline) from hHCN2. The amino acidsequence of the hHCN212 chimeric channel is set forth in SEQ ID NO: 2.This 889-amino acid long chimeric hHCN212 sequence shows 91.2% identitywith the 863-amino acid long mHCN212 sequence in 893 residues overlapwhen aligned for maximum correspondence.

FIG. 3. Amino Acid Sequence of the Mouse HCN212 Chimeric Channel.

The shaded N-terminal portion of the sequence is derived from mouseHCN2; the underlined intramembranous portion from mouse HCN1; and theC-terminal portion (without shading or underline) from mouse HCN2. Theamino acid sequence of the mouse HCN212 chimeric channel is set forth inSEQ ID NO: 6. This 863-amino acid long chimeric mHCN212 sequence shows91.2% identity with the 889-amino acid long hHCN212 sequence in 893residues overlap when aligned for maximum correspondence.

FIG. 4. A-D. Identification of Connexins in Gap Junctions of HumanMesenchymal Stem Cells (hMSCs).

Immunostaining of Cx43 (A), Cx40 (B) and Cx45 (C). D, Immunoblotanalysis of Cx43 in canine ventricle myocytes and hMSCs. Whole celllysates (120 μg) from ventricle cells or hMSCs were resolved by SDS,transferred to membranes, and blotted with Cx43 antibodies. Molecularweight markers are indicated.

FIG. 5. A-E. Macroscopic and Single Channel Properties of Gap JunctionsBetween hMSC Pairs.

Gap junction currents (k) elicited from hMSCs using a symmetricalbipolar pulse protocol (10 s, from ±10 mV to ±110 mV, V_(h)=0 mV) showedtwo types of voltage-dependent current deactivation: symmetrical (A) andasymmetrical (B). C, summary plots of normalized instantaneous (o) andsteady-state (•) g_(i) versus V_(j). Left panel, quasi-symmetricalrelationship from 5 pairs; continuous line, Boltzmann fit:V_(j,o)=−70/65 mV, g_(j,min)=0.29/0.34, g_(j,max)=0.99/1.00, z=2.2/2.3for negative/positive V_(j). Right panel, asymmetrical relationship from6 pairs; Boltzmann fit for negative V_(j): V_(j,0)=−72 mV,g_(j,min)=0.25, g_(j,max)=0.99, z=1.5. D and E, single channelrecordings from pairs of hMSCs. Pulse protocol (V₁ and V₂) andassociated multichannel currents (I₂) recorded from a cell pair duringmaintained V_(j) of ±80 mV. The discrete current steps indicate theopening andclosing of single channels. Dashed line: zero current level.The all points current histograms on the right-hand side reveal aconductance of ˜50 pS.

FIG. 6. A-E. Macroscopic Properties of Junctions in Cell Pairs Between ahMSC and HeLa Cell Expressing Only Cx40, Cx43 or Cx45.

In all cases hMSC to Hela cell coupling was tested 6 to 12 after hoursinitiating co-culture. A, Ij elicited in response to a series of 5-svoltage steps (V_(j)) in hMSC-HeLaCx43 pairs. Top, symmetrical currentdeactivation; bottom, asymmetrical current voltage dependence. B,Macroscopic Ij recordings from hMSC-HelaCx40 pairs exhibit symmetrical(top panel) and asymmetrical (bottom panel) voltage dependentdeactivation. C, Asymmetric Ij from hMSC-HeLaCx43 pair exhibits voltagedependent gating when Cx45 side is relatively negative. Ij recorded fromhMSC. D, g_(j,ss) plots versus V_(j) from pairs between hMSC andtransfected HeLa cells. Left panel, hMSC-HeLaCx43 pairs,quasi-symmetrical relationship (•) and asymmetrical relationship (o);continuous and dashed lines are Boltzmann fits (see text for details).Middle panel, symmetrical (•) and asymmetrical (o) relationships fromhMSC-HeLaCx40 pairs; the continuous and dashed lines correspond toBoltzmann fits (see text for details). Right panel, asymmetricalrelationship from hMSC-HeLaCx45 cell pairs; continuous line, Boltzmannfit for positive V_(j) (see text for details). E, Cell-to-cell LuciferYellow (LY) spread in cell pairs: from an hMSC to an hMSC (upper panel),from a HeLaCx43 to an hMSC (middle panel), and from an hMSC to aHeLaCx43 (bottom panel). In all cases a pipette containing 2 mM LY wasattached to the left-hand cell in the whole-cell configuration.Epifluorescent micrographs taken at 12 min after dye injection show LYspread to the adjacent (right-hand) cell. The simultaneously measuredjunctional conductance revealed gj of ˜13 nS, ˜16 nS, and ˜18 nS of thepairs, respectively. Cell Tracker green was used to distinguish hMSCsfrom HeLa cells or vice versa in all experiments.

FIG. 7. A-C. Macroscopic and Single Channel Properties of Gap JunctionsBetween hMSC-Canine Ventricle Cell Pairs.

Myocytes were plated between 12 and 72 h and co-cultured with hMSCs for6 to 12 h before measuring coupling. A, Localization of Cx43 forhMSC-canine ventricle cell pairs. Most of Cx43 was localized to theventricular cell ends and a small amount of Cx43 was present along thelateral borders. The intensive Cx43 staining was detected between theend of the rod-shaped ventricular cell (middle cell) and the hMSC (rightcell). There is no detectable Cx43 staining between the ventricular celland the hMSC on the left side. B, Top, phase-contrast micrograph of ahMSC-canine ventricular myocyte pair. Bottom, monopolar pulse protocol(V₁ and V₂) and associated macroscopic junctional currents (I₂)exhibiting asymmetrical voltage dependence. C, Top, multichannel currentelicited by symmetrical biphasic 60 mV pulse. Dashed line, zero currentlevel; dotted lines, represent discrete current steps indicative ofopening and closing of channels. The current histograms yielded aconductance of ˜40-50 pS. Bottom, multichannel recording duringmaintained V_(j) of 60 mV. The current histograms revealed severalconductances of 48-64 pS with several events with conductance of 84 pSto 99 pS (arrows) which resemble operation of Cx43, heterotypicCx40-Cx43 and/or homotypic Cx40 channels.

FIG. 8. A-B. Inactivation Properties of a Sodium Channel Suitable forIncorporation into a Cardiac Bypass Bridge.

The inactivation properties of the SKM-1 sodium channel measured inXenopus oocytes are shown. Left, (A) The holding potential ranged from−100 mV to −55 mV and the test potential was +30 mV. Right, (B) averagedinactivation curves for 11 oocytes.

FIG. 9. A-B. I-V Relationship of a Sodium Channel Suitable forIncorporation into a Cardiac Bypass Bridge.

Shown here is I-V relationship for the SKM-1 channel recorded in Xenopusoocytes. Left, (A) Raw data recorded from a holding potential of −80 mVto potentials between −70 mV and +50 mV in 10 mV increments. Right, (B)Peak inward 1-V relationship.

FIG. 10. Biophysical Properties of a Calcium Channel Suitable forIncorporation into a Cardiac Bypass Bridge.

CaV1.2, alpha2 and gamma, Pl.b and red fluorescent reporter wereco-expressed in HEK293 cells. Ca²⁺ current was recorded in the redfluorescent cells. The cells were held at −50 mV. Test pulses (60 ms)were from −40 mV to +50 mV with a 10 mV interval.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a bypass bridge comprising a tract of gapjunction-coupled cells having a first end and a second end, both endscapable of being attached to two selected sites in a heart so as toallow the conduction of a pacemaker and/or electrical signal/currentacross the tract between the two sites, wherein the cells functionallyexpress a sodium channel. In certain embodiments of the bypass bridge,cells in the tract further functionally express a pacemaker ion channelwhich induces a pacemaker current so as to induce a pacemaker current inthe cells. In preferred embodiments, the first endof the tract of cellsis capable of being attached to the atrium and the second end is capableof being attached to the ventricle, so as to form an atrioventricular(“AV”) bridge that allows conduction of a pacemaker and/or electricalsignal/current across the tract from the atrium to the ventricle andthereby stimulate contraction of the ventricle.

The invention also provides a bypass bridge comprising a tract of gapjunction-coupled cells having a first end and a second end, both endscapable of being attached to two selected sites in a heart so as toallow the conduction of a pacemaker and/or electrical signal/currentacross the tract between the two sites, wherein the cells functionallyexpress a potassium channel or calcium channel instead of a sodiumchannel. The embodiments disclosed herein for the bypass bridgeexpressing a sodium channel are equally applicable to the bypass bridgeexpressing a potassium or calcium channel.

Exemplary cells that may be used for growing the tract include, but arenot limited to, stem cells, cardiomyocytes, fibroblasts or skeletalmuscle cells engineered to express at least one cardiac connexin, orendothelial cells. In preferred embodiments, the stem cells are adultmesenchymal stem cell (MSCs) or embryonic stem cells (ESCs), whereinsaid stem cells are substantially incapable of differentiation. Invarious embodiments, the MSCs are autologous, allogeneic or heterogenicrelative to the subject into whose heart the bypass bridge is to beintroduced, and the subject is a human being. As used herein, a“subject” shall mean any animal or artificially modified animal. Animalsinclude, but are not limited to, humans, non-human primates, dogs, cats,cows, horses, sheep, pigs, rabbits, ferrets, rodents such as mice, ratsand guinea pigs, and birds such as chickens and turkeys. Artificiallymodified animals include, but are not limited to, SCID mice with humanimmune systems. In a preferred embodiment, the subject is a human.

In more preferred embodiments of the instant bypass bridge, the stemcells are human adult mesenchymal stem cells (hMSCs) or human embryonicstem cells (hESCs), wherein the stem cells are substantially incapableof differentiation. In other preferred embodiments, the hMSCs (a)express CD29, CD44, CD54, and HLA class I surface markers; and (b) donot express CD 14, CD34, CD45, and HLA class II surface markers. Inadditional embodiments, the hMSCs have been passaged at least 9 times,preferably 9-12 times. In further embodiments, the cells further expressat least one cardiac connexin. In still further embodiments, the atleast one cardiac connexin is Cx43, Cx40, or Cx45.

Thus, the present invention is based on the use of cell therapy toconstruct in the heart a bypass tract incorporating functional sodiumion channels, and optionally pacemaker channels, and optionally oralternatively potassium or calcium channels. The bypass bridge can beused to conduct a pacemaker and/or electrical signal/current between anytwo sites in the heart between which there is impaired pacemaker and/orelectrical conduction. For example, the bypass may be used as an AVbridge to take over or supplement the function of a diseased AV node.Human MSCs may be grown in culture on a non-bioreactive matrix into astrip or tract of cells containing two ends. Once growth is complete,one end of the tract may be attached, e.g., by sutures, to a firstselected site in the heart such as an atrium, and the other end mayattached to a second selected site such as a ventricle. In a bypassbridge used as an AV bridge, for example, pacemaker and/or electricalsignals/current generated by the sinus node to activate the atria willpropagate across the artificially constructed tract to excite theventricle. In this way the normal sequence of atrioventricularactivation will be maintained.

Human MSCs may be prepared in several ways including, but not limitedto, the following:

1: In culture without incorporation of additional molecular determinantsof conduction. Here the cells' own ability to form gap junctions thatcommunicate pacemaker and/or electrical signals are used as a means toconduct an electrotonic wave from one site in the heart to another.

2: In culture following transfection of the cells, such as byelectroporation or viral infection, to introduce a gene for at least oneof the cardiac connexins Cx43, Cx40 or Cx45, to enhance formation of gapjunctions and thereby facilitate cell-to-cell propagation ofpacemaker/electrical signals.

3: In culture following transfection of the cells to introduce a nucleicacid encoding the alpha and the accessory subunits of an L-type calciumchannel, thereby increasing the likelihood of not just electrotonicpropagation of a wavefront, but its active propagation by an actionpotential.

4: In culture following transfection of the cells to introduce a nucleicacid encoding the alpha subunit, with or without the accessory subunits,of a sodium channel, thereby increasing the likelihood of not justelectrotonic propagation or calcium-dependent propagation of awavefront, but its active propagation by a sodium-dependent actionpotential.

5: In culture following transfection of the cells to introduce a nucleicacid encoding the calcium and/or sodium channel subunits as in 3 and/or4, and a nucleic acid encoding an alpha subunit, with or without theaccessory subunits of a potassium channel, thereby increasing thelikelihood of not just active propagation by an action potential, butadditional control of the initial resting potential and its voltage-timecourse of repolarization and refractoriness.

6: Combinations of steps 2 through 5.

The preparation of a bypass bridge from hMSCs thus prepared allowsconduction of pacemaker and/or electrical signals/current between anytwo selected sites in the heart. In the case of an AV bridge, it willnot only facilitate propagation of signals from atrium to ventricle, butprovides sufficient delay from atrial to ventricular contraction tomaximize ventricular filling and emptying, thus mimicking the normalactivation and contractile sequence of the heart. Moreover, the cells inthe bypass bridge, preferably cells at one end of the tract, may also befurther transfected with a nucleic acid encoding a pacemaker currentchannel, wherein said transfected cells express a pacemaker current.Increases or decreases in calcium current, sodium current, potassiumcurrent or pacemaker current (I_(f)) may be obtained by increasing orreducing in the cells the expression of the ion channels carrying thesecurrents. These approaches, when used with gene therapy and stem celltechnology to improve atrial impulse initiation in the setting of sinusnode disease offer a completely physiologic system rather than itselectronic replacement. Thus, in embodiments of this invention, thebypass bridge expresses one or more genes encoding a pacemaker ionchannel and thereby induces a pacemaker current.

In different embodiments of this invention, the nucleic acid encodingthe ion channel or connexin gene is introduced into the cell byinfection with a viral vector, plasmid transformation, cosmidtransformation, electroporation, lipofection, transfection using achemical transfection reagent, heat shock transfection, ormicroinjection. In further embodiments, the viral vector is anadenoviral, an adeno-associated viral (AAV), or a retroviral vector.

In embodiments of the instant bypass bridge, the sodium channel is aSKM-1 channel. In certain embodiments, the SKM-1 channel comprises analpha subunit. In other embodiments, the SKM-1 channel further comprisesan accessory subunit. In additional embodiments, the tract furtherfunctionally expresses a potassium channel. The potassium channel maycomprise a Kir2. 1 or Kir2.2 alpha subunit, and may further comprise anaccessory subunit. In different embodiments of the bypass bridge, cellsin the tract further functionally express an L-type calcium channel,which may comprise an alpha subunit and accessory subunits. In furtherembodiments, cells in the tract forming the bypass bridge furtherfunctionally express one or more of at least one cardiac connexin, analpha subunit with accessory subunits of an L-type calcium channel, oran alpha subunit with or without accessory subunits of the potassiumchannel, so as to change the voltage-time course of repolarizationand/or refractoriness of the heart. In various embodiments, the at leastone cardiac connexin is Cx43, Cx40, or Cx45.

In various embodiments of the bypass bridge that expresses a pacemakerion channel, said pacemaker ion channel is at least one of (a) ahyperpolarization-activated, cyclic nucleotide-gated (HCN) ion channelor a mutant or chimera thereof, and (b) a MiRP1 beta subunit. The HCNchannel may be any of HCN1, HCN2, HCN3 or HCN4. In embodiments of thisinvention, the pacemaker channels are engineered to operate atprogressively slower rates, so as to equate to primary and subsidiarypacemakers similar to the sinus node and Purkinje system. In preferredembodiments, the pacemaker ion channel is expressed in cells in thefirst end of the tract. In more preferred embodiments, the cellsexpressing the pacemaker ion channel are located in a region extending0.5 mm from the first end.

Hyperpolarization-activated cation currents, termed I_(f), I_(h), orI_(q), were initially discovered in heart and nerve cells over 20 yearsago (for review, see DiFrancesco, 1993; Pape, 1996). These currents,carried by Na+ and K+ ions, contribute to a wide range of physiologicalfunctions, including cardiac and neuronal pacemaker activity, thesetting of resting potentials, input conductance and length constants,and dendritic integration (see Robinson and Siegelbaum, 2003; Biel etal., 2002). The hyperpolarization-activated, cyclic nucleotide-gated(HCN) family of ion channel subunits has been identified by molecularcloning (for review, see Clapham, 1998; Santoro and Tibbs, 1999; Biel etal., 2002), and when heterologously expressed, each of the fourdifferent HCN isoforms generates channels with the principal propertiesof native If, confirming that HCN channels are the molecular correlateof this current.

As used herein, a “HCN channel” shall mean ahyperpolarization-activated, cyclic nucleotide-gated ion channelresponsible for the hyperpolarization-activated cation currents that aredirectly regulated by cAMP and contribute to pacemaker activity in heartand brain. There are four HCN isoforms: HCN1, HCN2, HCN3 and HCN4. Allfour isoforms are expressed in brain; HCN1, HCN2 and HCN4 are alsoprominently expressed in heart, with HCN4 and HCN1 predominating insinoatrial node and HCN2 in the ventricular specialized conductingsystem. “mHCN” designates murine or mouse HCN; “hHCN” designates humanHCN.

HCN channels, similar to voltage-gatedK+ (Kv) channels, have foursubunits, each of which has six transmembrane segments, S1-S6: thepositively charged S4 domain forms the major voltage sensor, whereas S5and S6, together with the S5-S6 linker connecting the two, form the poredomain containing the ion permeation pathway and the gates that controlthe flow of ions (Larsson, 2002). Mutational studies on HCN channelsindicate that mutations in the S4 voltage sensor, the S4-S5 linkerimplicated in the coupling of voltage sensing to pore opening andclosing, the S5, S6 and S5-S6 linker which form the pore, the C-linker,and the C-terminal cyclic nucleotide binding domain (CNBD), may beparticularly important in affecting HCN channel activity. In embodimentsof the bypass bridge expressing a mutant HCN, said mutant HCN channelprovides an improved characteristic, as compared to a wild-type HCNchannel, selected from the group consisting of faster kinetics, morepositive activation, increased expression, increased stability,preserved or enhanced cAMP responsiveness, and preserved or enhancedneurohumoral response. Mutant HCN channels for inducing pacemakeractivity in cells is also described in U.S. Provisional Application Nos.60/781,723 (filed Mar. 14, 2006) and 60/832,515, entitled “Chimeric HCNChannels,” which is being filed concurrently with the subjectapplication (Jul. 21, 2006). Mutant HCN channels are also discussed inU.S. application Ser. No. 10/342,506. The preceding applications areherein incorporated by reference in their entirety.

In certain embodiments of the present invention, the mutant HCN channelcarries at least one mutation in S4 voltage sensor, the S4-S5 linker,S5, S6, the S5-S6 linker, and/or the C-linker, and the CNBD whichmutations result in one or more of the above discussed improvedcharacteristics. In other embodiments, the HCN mutant is E324A-HCN2,Y331A-HCN2, R339A-HCN2, or Y331A, E324A-HCN2. In preferred embodiments,the mutant HCN channel is E324A-HCN2.

In addition to the mutations noted above, many mutations in differentHCN isoforms have been reported. These include R318Q, W323A, E324A,E324D, E324K, E324Q, F327A, T330A and Y331A, Y331D, Y331F, Y331K, D332A,M338A, R339A, R339C, R339D, R339E and R339Q in HCN2 made by Chen et al.(2001a) to investigate in greater detail the role of the E324, Y331 and8339 residues in voltage sensing and activation. Chen et al. (2001b)have also reported the R538E and R591E mutations in mHCN1; Tsang et al.(2004) have reported G231A and M232A in mHCN1; Vemana et al (2004) havereported R247C, T249C, K250C, 1251C, L252C, S253C, L254C, L258C, R259C,L260C, S261C, C318S, S338C in mHCN2; Macri and Accili (2004) havereported S306Q, Y331D AND G404S in mHCN2; and Decher et al. (2004) havereported Y331A, Y331D, Y331S, R331FD, R339E, R339Q, 1439A, S441A, S441T,D443A, D443C, D443E, D443K, D443N, D443R, R447A, R447D, R447E, R447Y,Y449A, Y449D, Y449F, Y449G, Y449W, Y453A, Y453D, Y453F, Y453L, Y453W,P466Q, P466V, Y476A, Y477A and Y481A in mHCN2. The entire contents ofall of the above publications are incorporated herein by reference.Certain of the reported mutations listed above may confer, singly or incombination, beneficial characteristics on the HCN channel with regardto creating a biological pacemaker. The invention disclosed hereinencompasses all mutations in HCN channels, singly or in combinations,which improve pacemaker activity of the channel such as by providingfaster kinetics, more positive activation, increased expression and/orstability, preserved cyclic interval responsiveness, and/or preserved orenhanced neurohumoral response.

Mutations are identified herein by a designation with provides thesingle letter abbreviation of the amino acid residue that underwentmutation, the position of that residue within a polypeptide, and thesingle letter abbreviation of the amino acid residue to which theresidue was mutated. Thus, for example, E324A identifies a mutantpolypeptide in which the glutamate residue (E) at position 324 wasmutated to alanine (A). Y331A, E324A-HCN2 indicates a mouse HCN2 havinga double mutation, one in which tyrosine (Y) at position 331 was mutatedto alanine (A), and the other in which the glutamate residue at position324 was mutated to alanine.

In general terms, HCN polypeptides can be divided into three majordomains: (1) a cytoplasmic amino terminal domain; (2) the membranespanning domains and their linking regions; and (3) a cytoplasmiccarboxy-terminal domain. The N-terminal domain does not appear to play amajor role in channel activation (Biel et al., 2002). However, themembrane spanning domains with their linking regions play an importantrole in determining the kinetics of gating, whereas the CNBD is largelyresponsible for the ability of the channel to respond to the sympatheticand parasympathetic nervous systems that respectively raise and lowercellular cAMP levels.

In embodiments of the bypass bridge expressing a HCN chimera, thechimeric HCN channel preferably provides an improved characteristic, ascompared to a wild-type HCN channel, selected from the group consistingof faster kinetics, more positive activation, increased expression,increased stability, preserved or enhanced cAMP responsiveness, andpreserved or enhanced neurohumoral response. HCN chimeras for inducingpacemaker activity in cells is described in detail in U.S. ProvisionalApplication No. 60/715,934 (filed Sep. 9, 2005) and 60/832,515 entitled“Chimeric HCN Channels,” which is being filed concurrently with thesubject application (Jul. 21, 2006), both of which are hereinincorporated by reference in their entirety.

As used herein, a “HCN chimera” or “chimeric HCN channel” shall mean aHCN c channel comprising portions of more than one HCN channel isoform.Thus, a chimera may comprise portions of HCN1 and HCN2 or HCN3 or HCN4,and so forth. In preferred embodiments, the portions are an aminoterminal portion, an intramembranous portion, and a carboxy terminalportion. In other preferred embodiments, the portions are derived fromhuman HCN isoforms. In addition, an HCN chimera encompasses an ionchannel comprising portions of HCN channels derived from differentanimal species. Accordingly, in various embodiments of the bypassbridge, at least one portion of the HCN chimera is derived from ananimal species which is different from the animal species from which atleast one of the other two portions is derived. For example, one portionof the channel may be derived from a human and another portion may bederived from a non-human.

In additional embodiments of the instant bypass bridge, the HCN chimerais mHCN112, mHCN212, mHCN312, mHCN412, mHCN114, mHCN214, mHCN314,mHCN414, hHCN112, hHCN212, hHCN312, hHCN412, hHCN114, hHCN214, hHCN314,or hHCN414. In different embodiments, the HCN chimera is mHCN112,mHCN212, mHCN312, mHCN412, mHCN114, mHCN214, mHCN314, mHCN414, hHCN112,hHCN212, hHCN312, hHCN412, hHCN114, hHCN214, hHCN314, or hHCN414. In apreferred embodiment, the HCN chimera is hHCN212 having the sequence setforth in SEQ ID NO: 2 (see FIG. 2). In yet another preferred embodiment,the HCN chimera is mHCN212 having the sequence set forth in SEQ ID NO: 6(see FIG. 3).

As used herein, the term “HCNXYZ” (wherein X, Y and Z are any one of theintegers 1, 2, 3 or 4, with the proviso that at least one of X, Y and Zis a different number from at least one of the other numbers) shall meana chimeric HCN channel polypeptide comprising three contiguous portionsin the order XYZ, wherein X is an N-terminal portion, Y is anintramembrane portion, and Z is a C-terminal portion, and wherein thenumber X, Y or Z designates the HCN channel from which that portion isderived. For example, HCN112 is an HCN chimera with a N-terminal portionand intramembrane portion from HCN1 and a C-terminal portion from HCN2.

In certain embodiments of the bypass bridge, the HCN chimera comprisesan amino terminal portion contiguous with an intramembrane portioncontiguous with a carboxy terminal portion, wherein each portion is aportion of an HCN channel or a portion of a mutant thereof, and whereinone portion derives from an HCN channel or a mutant thereof which isdifferent from the HCN channel or mutant thereof from which at least oneof the other two portions derive. Thus, in various embodiments, at leastone portion of the chimera is derived from a HCN channel containing amutation which provides an improved characteristic, as compared to aportion from a wild-type HCN channel, selected from the group consistingof faster kinetics, more positive activation, increased expression,increased stability, preserved or enhanced cAMP responsiveness, andpreserved or enhanced neurohumoral response. In certain embodiments, themutant HCN channel contains a mutation in a region of the channelselected from the group consisting of the S4 voltage sensor, the S4-S5linker, SS, S6 and S5-S6 linker, the C-linker, and the CNBD. In otherembodiments, the mutant portion is derived from mHCN2 having thesequence set forth in SEQ ID NO: 14 (see FIG. 1) and comprisesE324A-mHCN2, Y331A-mHCN2, R339A-mHCN2, or Y331A, E324A-mHCN2. Inpreferred embodiments, the mutant portion comprises E324A-mHCN2.

The pacemaker activity of a HCN channel may be enhanced by co-expressingthe HCN channel with its beta subunit, MiRP 1, which increases themagnitude of the current expressed and/or speeds its kinetics ofactivation. See U.S. Pat. No. 6,783,979 and Qu et al. (2004), the entirecontents of which are incorporated herein by reference.

In certain embodiments of the bypass bridge disclosed herein, thepacemaker current is conducted by electrotonic conduction. In otherembodiments, the pacemaker current is actively propagated by an actionpotential. In further embodiments, the action potential is asodium-dependent action potential. In additional embodiments, cells inthe tract further functionally express an L-type calcium channel and theaction potential is a calcium-dependent action potential.

The present invention also provides a kit comprising a packagingmaterial containing therein (a) any of the bypass bridges disclosedherein. The kit may also contain labeling and instructions forimplanting the bypass bridge in a heart, so as to thereby treat adisorder associated with impaired conduction, or a disorder associatedwith both impaired conduction and impaired sinus node activity, in asubject's heart in a subject's heart.

This invention also provides a tandem pacemaker system comprising (1)any of the bypass bridges disclosed herein, and (2) an electronicpacemaker and/or a biological pacemaker, wherein the bypass bridgeoperates in tandem with the electronic and/or biological pacemaker tomore effectively treat a cardiac rhythm condition compared to the use ofthe biological or electronic pacemaker alone. In various embodiments,the bypass bridge is an AV bridge. The biological pacemaker orelectronic pacemaker may have a pacing level at, around, above or belowthe normal resting sinus node pacemaker level. In embodiments where abiological pacemaker or electronic pacemaker is used in tandem with anAV bridge described herein, the natural AV node may be ablated. Furtherdetails of tandem pacemaker systems comprising a bypass bridge may befound in U.S. Provisional Application Nos. 60/701,312 (filed Jul. 21,2005) and 60/781,723 (filed Mar. 14, 2006) and U.S. Ser. No. 11/490,997,filed concurrently herewith on Jul. 21, 2006, entitled “Tandem CardiacPacemaker System.” The proceeding applications are herein incorporatedby reference in their entirety.

The invention also provides a method of making a bypass bridge forimplantation in a heart comprising (a) transfecting a cell with, andfunctionally expressing therein, a nucleic acid encoding a sodiumchannel, and (b) growing the transfected cell into a tract of cellshaving a first and a second end capable of being attached to twoselected sites in the heart, wherein the cells are physicallyinterconnected via electrically conductive gap junctions. An embodimentof this method further comprises transfecting cells in the tract with anucleic acid encoding a pacemaker ion channel, wherein the nucleic acidis functionally expressed so as to induce a pacemaker current in thecells. In preferred embodiments, the pacemaker ion channel is expressedin cells in the first end of the tract. In more preferred embodiments,the cells expressing the pacemaker ion channel are located in a regionextending 0.5 mm from the first end. In these embodiments, thetransfection method used to introduce the pacemaker channel gene(s) tothe first end may be locally applied, or separated from the distalportions of the tract using physical barriers, in order to restricttransfection to first end portion.

In various embodiments, the pacemaker ion channel is at least one of (a)a hyperpolarization-activated, cyclic nucleotide-gated (HCN) ion channelor a mutant or chimera thereof, and (b) a MiRP 1 beta subunit. Inpreferred embodiments of the instant methods, the cells are hMSCs orhESCs, wherein said cells are substantially incapable ofdifferentiation. In further embodiments, the human adult mesenchymalstem cells (a) express CD29, CD44, CD54, and HLA class I surfacemarkers; and (b) do not express CD14, CD34, CD45, and HLA class IIsurface markers. In still further embodiments, the human adultmesenchymal stem cells have been passaged at least 9 times.

Additional embodiments of the instant methods for making a bypass bridgefurther comprise transfecting the cell with, and expressing therein, atleast one nucleic acid encoding one or more of at least one cardiacconnexin, an alpha subunit with accessory subunits of an L-type calciumchannel, or an alpha subunit with or without accessory subunits of thepotassium channel, such that implantation of a bypass bridge in a heartchanges the voltage-time course of repolarization and/or refractorinessof the heart.

Variations of the instant methods for making a bypass bridge comprise(a) growing a cell into a tract of cells having a first and a second endcapable of being attached to two selected sites in the heart, whereinthe cells are physically interconnected via electrically conductive gapjunctions, before (b) transfecting the cells in the tract with, andfunctionally expressing therein, a nucleic acid encoding a sodiumchannel, and optionally other ion channel genes and/or a pacemakerchannel gene.

This invention further provides a method of implanting a bypass bridgein a heart comprising (a) making a bypass bridge comprising a tract ofcells by any of the methods disclosed herein, (b) selecting a first anda second site in the heart, and (c) attaching the first end of the tractto the first site and the second end of the tract to the second site, soas to thereby implant a bypass bridge in the heart that allows theconduction of a pacemaker and/or electrical signal/current across thetract between the two sites. Other embodiments further comprisetransfecting cells in the tract with a nucleic acid encoding a pacemakerion channel, wherein the nucleic acid is functionally expressed so as toinduce a pacemaker current in the cells. In preferred embodiments, thepacemaker ion channel is expressed in cells in the first end of thetract. In more preferred embodiments, the cells expressing the pacemakerion channel are located in a region extending 0.5 mm from the first end.In other preferred embodiments, the first site is in an atrium and thesecond site is in a ventricle, so as to allow propagation of a pacemakerand/or electrical signal/current across the tract from the atrium to theventricle. In different embodiments of the instant methods, the apacemaker and/or electrical signal/current is generated in the atrium bythe sinus node, an electronic pacemaker, a biological pacemaker, orcells within the bypass bridge expressing a pacemaker current. Infurther embodiments, the pacemaker ion channel is at least one of (a) ahyperpolarization-activated, cyclic nucleotide-gated (HCN) ion channelor a mutant or chimera thereof, and (b) a MiRP1 beta subunit.

In additional embodiments of the instant methods, the cells in the tractof the bypass bridge are stem cells, cardiomyocytes, fibroblasts orskeletal muscle cells engineered to express at least one cardiacconnexins, or endothelial cells. In various embodiments, the stem cellsare adult MSCs or ESCs, wherein said cells are substantially incapableof differentiation. In preferred embodiments, the stem cells are hMSCsor hESCs, wherein said stem cells are substantially incapable ofdifferentiation. Other embodiments further comprise transfecting thecells with, and expressing therein, at least one nucleic acid encodingone or more of at least one cardiac connexin, an alpha subunit withaccessory subunits of an L-type calcium channel, or an alpha subunitwith or without accessory subunits of the potassium channel, so as tochange the voltage-time course of repolarization and/or refractorinessof the heart. In further embodiments, the at least one connexin is Cx43,Cx40, or Cx45.

The present invention also provides a method of treating a disorderassociated with an impaired conduction in a subject's heart comprising(a) transfecting a cell with a nucleic acid encoding a sodium channel,wherein the cell functionally expresses the sodium channel, (b) growingthe transfected cell into a tract of cells having a first end and asecond end, wherein the cells are physically interconnected viaelectrically conductive gap junctions, (c) selecting a first site and asecond site in the subject's heart between which sites conduction isimpaired, and (d) attaching the first end of the tract to the first siteand the second end of the tract to the second site, so as to allow theconduction of a pacemaker and/or electrical signal/current across thetract between the two sites and thereby treat the subject.

As used herein, “treating” a disorder shall mean causing the subjectafflicted with the disorder to experience a reduction, remission orregression of the disorder and/or its symptoms. In various embodiments,recurrence of the disorder and/or its symptoms is prevented. Inpreferred embodiments, the subject is cured of the disorder and/or itssymptoms. To “functionally express” a nucleic acid shall mean that acell or other biological system into which the nucleic acid has beenintroduced produces a functional polypeptide encoded by the nucleicacid. The encoded polypeptide itself may also be said to be functionallyexpressed.

This invention further provides a method of treating a disorderassociated with an impaired conduction and impaired sinus node activityin a subject's heart comprising (a) transfecting a cell with at leastone nucleic acid encoding a sodium channel and a pacemaker ion channel,wherein the cell functionally expresses the sodium channel and thepacemaker ion channel, (b) growing the transfected cell into a tract ofcells having a first end and a second end, wherein the cells arephysically interconnected via electrically conductive gap junctions, (c)selecting a first site in the left atrium of the heart and a secondsite, between which sites conduction is impaired, and (d) attaching thefirst end of the tract to the first site and the second end of the tractto the second site, so as to allow the propagation of a pacemaker and/orelectrical signal/current generated by the sinus node and/or tract ofcells between the two sites and thereby treat the subject.

In embodiments of the instant methods, the cells are hMSCs or hESCs,wherein said stem cells are substantially incapable of differentiation.Various embodiments further comprise transfecting the cells in the tractwith, and expressing therein, at least one nucleic acid encoding one ormore of at least one cardiac connexin, an alpha subunit with accessorysubunits of an L-type calcium channel, or an alpha subunit with orwithout accessory subunits of the potassium channel, so as to change thevoltage-time course of repolarization and/or refractoriness of theheart. In further embodiments, the pacemaker ion channel is at least oneof (a) a hyperpolarization-activated, cyclic nucleotide-gated (HCN) ionchannel or a mutant or chimera thereof, and (b) a MiRP1 beta subunit.

The present invention encompasses a variety of kits comprising thebypass bridge of the instant invention, as well as methods for making abypass bridge, methods of implanting a bypass bridge in a heart, andmethods of treating a disorder, corresponding to each of the differentembodiments of the bypass bridge disclosed herein. These variant kitsand methods are not all individually described in detail herein but willbe readily evident and understood to one of ordinary skill in the artbased on the disclosure of the different embodiments of the bypassbridge. Accordingly, the various embodiments described herein may beequally applied as appropriate to the bypass bridges, kits and methodsof this invention.

The following Examples are presented to aid in understanding theinvention, and are not intended, and should not be construed, to limitin any way the invention set forth in the claims which followthereafter. These Examples do not include detailed descriptions ofexperimental methods that are well known to those of ordinary skill inthe art, such as methods used in the construction of recombinant nucleicacid vectors, transfection of host cells with such recombinant vectors,and the functional expression of genes in transfected cells. Detaileddescriptions of such conventional methods are provided in numerouspublications, including Sambrook et al. (1989), the contents of whichare hereby incorporated herein in their entirety.

Example 1 Culture of Gap Junction-Coupled Human Mesenchymal Stem Cells

Cell Cultures

Human mesenchymal stem cells (hMSCs; mesenchymal stem cells, human bonemarrow; Poietics™) were purchased from Clonetics/BioWhittaker(Walkersville, Md., USA), cultured in mesenchymal stem cell (MCS) growthmedium and used from passages 2-4. Isolated and purified hMSCs can becultured for many passages (12) without losing their unique properties,i.e., normal karyotype and telomerase activity (van den Bos et al.,1997; Pittenger et al., 1999).

HeLa cells transfected with rat Cx40, rat Cx43 or mouse Cx45 werecocultured with hMSCs. Production, characterization and cultureconditions of transfected HeLa cells have been previously described(Elfgang et al., 1995; Valiunas et al., 2000; 2002).

Anti-Connexin Antibodies, Immunofluorescent Labeling, and ImmunoblotAnalysis

Commercially available mouse anticonnexin monoclonal and polyclonalantibodies (Chemicon International, Temecula, Calif.) of Cx40, Cx43 andCx45 were used for immunostaining and immunoblots as described earlier(Laing and Beyer, 1995). Fluorescein-conjugated goat antimouse orantirabbit IgG (ICN Biomedicals, Inc., Costa Mesa, Calif.) was used assecondary antibody.

Electrophysiological Measurements Across Gap Junctions

Glass coverslips with adherent cells were transferred to an experimentalchamber perfused at room temperature (˜22° C.) with bath solutioncontaining (mM): NaCl, 150; KCl, 10; CaCl2, 2; Hepes, 5 (pH 7.4);glucose, 5. The patch pipettes were filled with solution containing(mM): potassium aspartate, 120; NaCl, 10; MgATP, 3; Hepes, 5 (pH 7.2);EGTA, 10 (pCa ˜8); filtered through 0.22 μm pores. When filled, theresistance of the pipettes measured 1-2 MΩ. Experiments were carried outon cell pairs using a double voltage-clamp. This method permittedcontrol of the membrane potential (Vm) and measurement of the associatedjunctional currents (Ij).

Dye Flux Studies

Dye transfer through gap junction channels was investigated using cellpairs. Lucifer Yellow (LY; Molecular Probes) was dissolved in thepipette solution to reach a concentration of 2 mM. Fluorescent dyecell-to-cell spread was imaged using a 16 bit 64 000 pixel grey scaledigital CCD-camera (LYNXX 2000T, SpectraSource Instruments, WestlakeVillage, Calif.) (Valiunas et al., 2002). In experiments withheterologous pairs, LY was always injected into the cells which weretagged with Cell Tracker Green. The injected cell fluorescence intensityderived from LY is 10-15 times higher than the initial fluorescence fromCell Tracker Green.

Human MSCs Express Connexins

The connexins, Cx43 and Cx40, were immunolocalized, as evidenced bytypical punctate staining, along regions of intimate cell-to-cellcontact and within regions of the cytoplasm of the hMSCs grown inculture as monolayers (FIGS. 4A, B). Cx45 staining was also detected,but unlike that of Cx43 or Cx40, was not typical of connexindistribution in cells. Rather, it was characterized by fine granularcytoplasmic and reticular-like staining with no readily observedmembrane-associated plaques (FIG. 4C). This does not exclude thepossibility that Cx45 channels exist but does imply that their numberrelative to Cx43 and Cx40 homotypic, heterotypic and heteromericchannels is low. FIG. 4D illustrates Western blot analysis for canineventricle myocytes and hMSCs with a Cx43 polyclonal antibody which addsfurther proof of Cx43 presence in hMSCs.

Gapjunctional Coupling Between hMSCs and Various Cell Lines

Gap junctional coupling among hMSCs is demonstrated in FIG. 5.Junctional currents recorded between hMSC pairs show quasi-symmetrical(FIG. 5A) and asymmetrical (FIG. 5B) voltage dependency arising inresponse to symmetrical 10-s transjunctional voltage steps (V_(j)) ofequal amplitude but opposite sign starting from ±10 mV to ±110 mV usingincrements of 20 mV. These behaviors are typically observed in cellswhich co-express Cx43 and Cx40 (Valiunas et al., 2001).

FIG. 5C summarizes the data obtained from hMSC pairs. The values ofnormalized instantaneous (g_(j,inst), o) and steady state conductances(g_(j,ss), •) (determined at the beginning and at the end of each V_(j)step, respectively) were plotted versus V_(j). The left panel shows aquasi-symmetrical relationship from five hMSC pairs. The continuouscurves represent the best fit of data to the Boltzmann equation with thefollowing parameters: half-deactivation voltage, V_(j,o)=−70/65 mV;minimum g_(j), g_(j,min)=0.29/0.34; maximum g_(j), g_(j),max=0.99/1.00;gating charge, z=2.2/2.3 for negative/positive V, respectively.Summarized plots from six asymmetrical cases are shown in the rightpanel. The g_(j,ss) declined in sigmoidal fashion at negative V_(j) andshowed a reduced voltage sensitivity to positive V_(j). Boltzman fittingfor negative V_(j) revealed the following values: V_(j,o)=−72 mV,g_(j,min)=0.25, g_(j,max)=0.99, z=1.5.

FIGS. 5D and E illustrate typical multichannel recordings from a hMSCpair. Using 120 mM K aspartate as a pipette solution, channels wereobserved with unitary conductances of 28-80 pS range. Operation ofchannels with ˜50 pS conductance (see FIG. 5D) is consistent withpreviously published values (Valiunas et al., 1997; 2002) for Cx43homotypic channels. This does not preclude the presence of other channeltypes, it merely suggests that Cx43 forms functional channels in hMSCs.

To further define the nature of the coupling, hMSCs were co-culturedwith human HeLa cells stably transfected with Cx43, Cx40, and Cx45(Elfgang et al., 1995) and it was found that hMSCs were able to coupleto all these transfectants. FIG. 6A shows an example of junctionalcurrents recorded between an hMSC and HeLaCx43 cell pairs thatmanifested symmetrically and asymmetrically voltage dependent currentsin response to a series (from ±10 mV to ±110 mV) of symmetricaltransjunctional voltage steps (V_(j)). The quasi-symmetric recordsuggests that the dominant functional channel is homotypic Cx43 whilethe asymmetric record suggests the activity of another connexin in thehMSC (presumably Cx40 as shown by immunohistochemistry, see FIG. 4) thatcould be either a heterotypic or heteromeric form or both. These recordsare similar to those published for transfected cells: heterotypic andmixed (heteromeric) forms of Cx40 and Cx43 (Valiunas et al., 2000;2001). Co-culture of hMSCs with HeLa cells transfected with Cx40 (FIG.6B) also revealed symmetric and asymmetric voltage dependent junctionalcurrents consistent with the co-expression of Cx43 and Cx40 in the hMSCssimilar to the data for Cx43 HeLa-hMSC pairs. HeLa cells transfectedwith Cx45 coupled to hMSCs always produced asymmetric junctionalcurrents with pronounced voltage gating when Cx45 (HeLa) side wasnegative (FIG. 6C). This is consistent with the dominant channel formsin the hMSC being Cx43 and Cx40 as both produce asymmetric currents whenthey form heterotypic channels with Cx45 (Valiunas et al., 2000; 2001).This does not exclude Cx45 as a functioning channel in hMSCs but it doesindicate that Cx45 is a minor contributor to cell to cell coupling inhMSCs. The lack of visualized plaques in the immunostaining for Cx45(FIG. 7) further supports this interpretation.

The summarized plots of g_(j,ss) versus V_(j) from pairs between hMSCand transfected HeLa cells are shown in FIG. 6D. The left panel showsthe results from hMSC-HeLaCx43 pairs. For symmetrical data (•, fourpreparations), Boltzmann fits (continuous lines) yielded the followingparameters: V_(j,0)=−61/65 mV, g_(j,min)=0.24/0.33, g_(j,max)=0.99/0.99,z=2.4/3.8 for negative/positive Vj. For asymmetrical data (o, threepreparations), the Boltzmann fit (dashed line) at negative _(Vj) valuesrevealed the following parameter values: V_(j,o)=−70 mV, g_(j,min)=0.31,g_(j,max)=1.00, z=2.2. The middle panel shows data from hMSC-HeLaCx40pairs including three symmetrical (•) and two asymmetrical (o)g_(j,ss)−V_(j)-relationships. The continuous lines correspond to aBoltzmann fit to symmetrical data (V_(j,o)=−57/76 mV,g_(j,min)=0.22/0.29, g_(j,max)=1.1/1.0, z=1.4/2.3; negative/positive Vj)and the dashed line is a fit to the asymmetrical data (Vj,o=−57/85 mV,gj,min=0.22/0.65, g_(j,max)=1.1/1.0, z=1.3/2.2; negative/positiveV_(j)). The data from the six complete experiments from hMSC-HeLaCx45cell pairs are shown on the right panel. The g_(j,ss) plot versus V_(j)was strongly asymmetrical and the best fit of the data to the Boltzmannequation at positive V_(j) values revealed following parameter values:V_(j,0)=31 mV, g_(j,min)=0.07, g_(j,max)=1.2, z=1.8.

FIG. 6E shows Lucifer Yellow transfer from an hMSC to an hMSC (upperpanel), from a HeLaCx43 to an hMSC (middle panel), and from an hMSC to aHeLaCx43 (bottom panel). The junctional conductance of the cell pairswas simultaneously measured by methods described earlier (Valiunas etal., 2002) and revealed conductances of ˜13, ˜16 and ˜18 nS,respectively. The transfer of Lucifer Yellow was similar to thatpreviously reported for homotypic Cx43 or co-expressed Cx43 and Cx40 inHeLa cells (Valiunas et al., 2002). Cell Tracker Green (MolecularProbes) was always used in one of the two populations of cells to allowheterologous pairs to be identified (Valiunas et al., 2000). LuciferYellow was always delivered to the cell containing cell tracker. Thefluorescence intensity generated by the Cell Tracker Green was 10-15times less than fluorescence intensity produced by the concentration ofLucifer Yellow delivered to the source cell.

Human MSCs were also co-cultured with adult canine ventricular myocytesas shown in FIG. 7. Immunostaining for Cx43 was detected between therod-shaped ventricular myocytes and hMSCs as shown in FIG. 7A. The hMSCscouple electrically with cardiac myocytes. Both macroscopic (FIG. 7B)and multichannel (FIG. 7C) records were obtained. Junctional currents inFIG. 7B are asymmetrical while those in FIG. 7C show unitary events ofthe size range typically resulting from the operation of homotypic Cx43or heterotypic Cx43-Cx40 or homotypic Cx40 channels (Valiunas et al.,2000; 2001). Heteromeric forms are also possible whose conductances arethe same or similar to homotypic or heterotypic forms.

The studies of cell pairs have demonstrated effective coupling of hMSCto other hMSC (13.8±2.4 nS, n=14), to HeLaCx43 (7.9±2.1 nS, n=7), toHeLaCx40 (4.6±2.6 nS, n=5), to HeLaCx45 (11±2.6 nS, n=5.), and toventricular myocytes (1.5±1.3 nS, n=4).

These data suggest that MSCs should readily integrate into electricalsyncytia of many tissues, promoting repair or serving as the substratefor a therapeutic delivery system. In particular, the data support thepossibility of using hMSCs as a therapeutic substrate for repair ofcardiac tissue. Other syncytia such as vascular smooth muscle orendothelial cells should also be able to couple to the hMSCs because ofthe ubiquity of Cx43 and Cx40 (Wang et al., 2001; Beyer, 1993). Thus,they may also be amenable to hMSCs-based therapeutics. For example,hMSCs can be transfected to express ion channels which then caninfluence the surrounding synctial tissue. Alternatively, the hMSCs canbe transfected to express genes that produce small therapeutic moleculescapable of permeating gap junctions and influencing recipient cells.Further, for short term therapy, small molecules can be directly loadedinto hMSCs for delivery to recipient cells. The success of suchapproaches is dependent on gap junction channels as the final conduitfor delivery of the therapeutic agent to the recipient cells. Thefeasibility of the first approach has been demonstrated herein bydelivering HCN2-transfected hMSCs to the canine heart where theygenerate a spontaneous rhythm

Example 2 Ion channels Suitable for Incorporation into a Cardiac BypassBridge

As previously described herein, hMSCs form gap junctions that permit atract of physically connected cells to conduct electrical signals byelectrotonic conduction. Cell-to-cell propagation of electrical signalsmay be facilitated by functionally expressing in the cells one or morenucleic acids encoding at least one of the cardiac connexins Cx43, Cx40or Cx45 in order to enhance formation of gap junctions. The expressionin the cells of a nucleic acid(s) encoding the alpha subunit, with orwithout the accessory subunits, of a sodium channel, or the alpha andaccessory subunits of an L-type calcium channel, also increases thelikelihood of not just electrotonic propagation of a wavefront, but itsactive propagation by a sodium-dependent or calcium-dependent actionpotential. In addition, expression of a potassium channel in the cellsboth increases the likelihood of active propagation by an actionpotential and provides a means of controlling the initial restingpotential and its voltage-time course of repolarization andrefractoriness.

The biophysical properties of a SKM-1 sodium channel and a L-typecalcium channel were assayed to investigate their suitability forproviding an action potential in acardiac bypass bridge. Theinactivation properties of the SKM-1 sodium channel measured in Xenopusoocytes are shown in FIG. 8, and the 1-V relationship for the SKM-1channel in Xenopus oocytes is shown in FIG. 9. The CaV1.2, alpha2&gamma,Pl.b and red fluorescent reporter were co-expressed in HEK293 cells.FIG. 10 shows the Ca2+ current recorded in the red fluorescent cells.

The SKM-1 sodium channel and L-type calcium channel are used to“fine-tune” the conductivity of a bypass bridge to the extent that thisis needed. One embodiment of the bypass bridge described herein is an AVbridge. Incorporation of sodium channels in an AV bridge can be viewedas counterintuitive in that the sodium channel is usually associatedwith very rapid conduction, whereas an AV bypass should conduct moreslowly to operate most efficiently. However, because the degree ofcoupling among the cells can be manipulated it is possible to use achannel that routinely propagates rapidly and expect this to conductmore slowly. Incorporation of calcium channels, for inward calciumcurrent, will elevate the plateau of the cardiac action potential andprolong repolarization and refractoriness. In addition, functionallyexpressing potassium channels would accelerate repolarization and canthus shorten refractoriness. Therefore, by manipulating the sodium,calcium and potassium currents and cell coupling, the characteristics ofthe cardiac electrical activity can be correspondingly manipulated.

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1. An atrioventricular bypass bridge comprising a tract of gapjunction-coupled adult human mesenchymal stem cells obtained from blood,wherein the tract i. is formed in vitro, ii. is at least about 0.5 mm inlength, iii. has a first end that is capable of being attached to afirst site in the atrium of the heart and a second end that is capableof being attached to a second site in the ventricle of the heart so asto allow the conduction of at least one of a pacemaker signal, pacemakercurrent, electrical signal, and electrical current across the tractbetween the two sites, and iv. wherein the adult mesenchymal stem cellsfunctionally express both a sodium channel and a potassium channel whichchannels are each encoded by a respective nucleic acid that has beenintroduced into the cells.
 2. The bypass bridge of claim 1, wherein themesenchymal stem cells further functionally express a pacemaker ionchannel encoded by a nucleic acid that has been introduced into thecells which channel induces a pacemaker in the cells. 3-6. (canceled) 7.The bypass bridge of claim 1 or 2, wherein the sodium channel is a SKM-1channel.
 8. The bypass bridge of claim 7, wherein the SKM-1 channelcomprises an alpha subunit.
 9. The bypass bridge of claim 8, wherein theSKM-1 channel further comprises an accessory subunit.
 10. The bypassbridge of claim 1, wherein the cells in the tract further functionallyexpress a calcium channel encoded by a nucleic acid that has beenintroduced into the cells.
 11. The bypass bridge of claim 1, wherein thepotassium channel comprises the potassium inwardly-rectifying channel 12(Kir2.1) alpha subunit or potassium inwardly-rectifying channel,subfamily 1, member 12 (Kir2.2) alpha subunit.
 12. The bypass bridge ofclaim 11, wherein the potassium channel further comprises an accessorysubunit.
 13. The bypass bridge of claim 10, wherein the calcium channelis an L-type calcium channel.
 14. The bypass bridge of claim 13, whereinthe L-type calcium channel comprises an alpha subunit and accessorysubunits.
 15. The bypass bridge of claim 1, wherein cells in the tractfurther functionally express one or more of at least one cardiacconnexin, an alpha subunit with accessory subunits of an L-type calciumchannel, or an alpha subunit with or without accessory subunits of thepotassium channel.
 16. The bypass bridge of claim 15, wherein the atleast one connexin is Cx43, Cx40, or Cx45.
 17. The bypass bridge ofclaim 2, wherein the pacemaker ion channel is at least one of (a) ahyperpolarization-activated, cyclic nucleotide-gated (HCN) ion channelor chimera thereof, and (b) a MiRP1 beta subunit.
 18. The bypass bridgeof claim 17, wherein the pacemaker ion channel is expressed in cells inthe first end of the tract.
 19. The bypass bridge of claim 18, whereinthe cells expressing the pacemaker ion channel are located in a regionextending 0.5 mm from the first end.
 20. The bypass bridge of claim 17,wherein the chimeric HCN channel provides an improved characteristic, ascompared to a wild-type HCN channel, selected from the group consistingof faster kinetics, more positive activation, increased expression,increased stability, preserved or enhanced cAMP responsiveness, andpreserved or enhanced neurohumoral response.
 21. The bypass bridge ofclaim 17, wherein the HCN chimera comprises portions derived from morethan one HCN channel isoform.
 22. The bypass bridge of claim 21, whereinthe portions are an amino terminal portion, an intramembranous portion,and a carboxy terminal portion.
 23. The bypass bridge of claim 21,wherein at least one portion of the HCN chimera is derived from ananimal species which is different from the animal species from which atleast one of the other two portions is derived.
 24. The bypass bridge ofclaim 21, wherein the HCN chimera is mHCN112, mHCN212, mHCN312, mHCN412,mHCN114, mHCN214, mHCN314, mHCN414, hHCN112, hHCN212, hHCN312, hHCN412,hHCN114, hHCN214, hHCN314, or hHCN414.
 25. The bypass bridge of claim24, wherein the HCN chimera is hHCN212 having the sequence set forth inSEQ ID NO:
 2. 26. The bypass bridge of claim 24, wherein the HCN chimerais mHCN212 having the sequence set forth in SEQ ID NO:
 6. 27. The bypassbridge of claim 21, wherein at least one portion of the chimera isderived from a HCN channel containing a mutation which provides animproved characteristic, as compared to a wild-type HCN channel,selected from the group consisting of faster kinetics, more positiveactivation, increased expression, increased stability, preserved orenhanced cAMP responsiveness, and preserved or enhanced neurohumoralresponse.
 28. The bypass bridge of claim 17, wherein the HCN channel isa mutant channel derived from mHCN2 having the sequence set forth in SEQID NO: 14 and comprises E324A-mHCN2, Y331A-mHCN2, R339A-mHCN2, or Y331A,E324A-mHCN2.
 29. The bypass bridge of claim 28, wherein the mutant HCNchannel is E324A-HCN2.
 30. The bypass bridge of claim 19, wherein thepacemaker current is conducted by electrotonic conduction.
 31. Thebypass bridge of claim 17, wherein the pacemaker current is activelypropagated by an action potential.
 32. The bypass bridge of claim 31,wherein the action potential is a sodium-dependent action potential. 33.The bypass bridge of claim 31, wherein cells in the tract furtherfunctionally express an L-type calcium channel and the action potentialis a calcium-dependent action potential.
 34. A method of making a bypassbridge for implantation in a heart comprising: (a) transfecting a cellwith, and functionally expressing therein, a nucleic acid encoding asodium channel; and (b) growing the transfected cell into a tract ofcells having a first and a second end capable of being attached to twoselected sites in the heart, wherein the cells are physicallyinterconnected via electrically conductive gap junctions.
 35. The methodof claim 34 for making a bypass bridge, further comprising transfectingcells in the tract with a nucleic acid encoding a pacemaker ion channel,wherein the nucleic acid is functionally expressed so as to induce apacemaker current in the cells.
 36. The method of claim 34 or 35,wherein the cells are human adult mesenchymal stem cells.
 37. The methodof claim 34 or 35, further comprising transfecting the cell with, andexpressing therein, at least one nucleic acid encoding one or more of atleast one cardiac connexin, an alpha subunit with accessory subunits ofan L-type calcium channel, or an alpha subunit with or without accessorysubunits of the potassium channel, such that implantation of a bypassbridge in a heart changes the voltage-time course of repolarizationand/or refractoriness of the heart.
 38. The method of claim 41, whereinthe pacemaker ion channel is at least one of (a) ahyperpolarization-activated, cyclic nucleotide-gated (HCN) ion channelor a mutant or chimera thereof, and (b) a MiRP1 beta subunit.
 39. Amethod of implanting a bypass bridge in a heart comprising: (a) making abypass bridge by the method of claim 34; (b) selecting a first and asecond site in the heart; and (c) attaching the first end of the tractto the first site and the second end of the tract to the second site; soas to thereby implant a bypass bridge in the heart that allows theconduction of a pacemaker and/or electrical signal/current across thetract between the two sites.
 40. The method of claim 39, wherein the apacemaker and/or electrical signal/current is generated in the atrium bythe sinus node or an electronic pacemaker.
 41. The method of claim 39,further comprising transfecting cells in the tract with a nucleic acidencoding a pacemaker ion channel, wherein the nucleic acid isfunctionally expressed so as to induce a pacemaker current in the cells.42. The method of claim 41, wherein the pacemaker ion channel isexpressed in cells in the first end of the tract.
 43. The method ofclaim 42, wherein the cells expressing the pacemaker ion channel arelocated in a region extending 0.5 mm from the first end.
 44. The methodof claim 39 or 41, wherein the first site is in an atrium and the secondsite is in a ventricle, so as to allow propagation of a pacemaker and/orelectrical signal/current across the tract from the atrium to theventricle.
 45. The method of claim 39 or 41, wherein the cells are stemcells, cardiomyocytes, fibroblasts or skeletal muscle cells engineeredto express connexins, or endothelial cells.
 46. The method of claim 44,wherein the stem cells are adult mesenchymal stem cells or embryonicstem cells, wherein said cells are substantially incapable ofdifferentiation.
 47. The method of claim 46, wherein the stem cells arehuman adult mesenchymal stem cells or human embryonic stem cells. 48.The method of claim 39 or 41, further comprising transfecting the cellsin the tract with, and expressing therein, at least one nucleic acidencoding one or more of at least one cardiac connexin, an alpha subunitwith accessory subunits of an L-type calcium channel, or an alphasubunit with or without accessory subunits of the potassium channel, soas to change the voltage-time course of repolarization and/orrefractoriness of the heart.
 49. The method of claim 48, wherein the atleast one cardiac connexin is Cx43, Cx40, or Cx45.
 50. The method ofclaim 41, wherein the pacemaker ion channel is at least one of (a) ahyperpolarization-activated, cyclic nucleotide-gated (HCN) ion channelor a mutant or chimera thereof, and (b) a MiRP1 beta subunit.
 51. Amethod of treating a disorder associated with an impaired conduction ina subject's heart comprising: (a) transfecting a cell with a nucleicacid encoding a sodium channel, wherein the cell functionally expressesthe sodium channel; (b) growing the transfected cell into a tract ofcells having a first end and a second end, wherein the cells arephysically interconnected via electrically conductive gap junctions; (c)selecting a first site and a second site in the heart between whichsites conduction is impaired; and (d) so as to allow the conduction of apacemaker and/or electrical signal/current across the tract between thetwo sites and thereby treat the subject.
 52. A method of treating adisorder associated with an impaired conduction and impaired sinus nodeactivity in a subject's heart comprising: (a) transfecting a cell withat least one nucleic acid encoding a sodium channel and a pacemaker ionchannel, wherein the cell functionally expresses the sodium channel andthe pacemaker ion channel; (b) growing the transfected cell into a tractof cells having a first end and a second end, wherein the cells arephysically interconnected via electrically conductive gap junctions; (c)selecting a first site in the left atrium of the heart and a secondsite, between which sites conduction is impaired; and (d) attaching thefirst end of the tract to the first site and the second end of the tractto the second site; so as to allow the propagation of a pacemaker and/orelectrical signal/current generated by the sinus node and/or tract ofcells between the two sites and thereby treat the subject.
 53. Themethod of claim 51 or 52, wherein the cells are human adult mesenchymalstem cells or human embryonic stem cells, wherein said cells aresubstantially incapable of differentiation.
 54. The method of claim 51or 52, further comprising transfecting the cells with, and expressingtherein, at least one nucleic acid encoding one or more of at least oneconnexin, an alpha subunit with accessory subunits of an L-type calciumchannel, or an alpha subunit with or without accessory subunits of thepotassium channel, so as to change the voltage-time course ofrepolarization and/or refractoriness of the heart.
 55. The method ofclaim 52, wherein the pacemaker ion channel is at least one of (a) ahyperpolarization-activated, cyclic nucleotide-gated (HCN) ion channelor a mutant or chimera thereof, and (b) a MiRP1 beta subunit.
 56. Anatrioventricular bypass tract made by growing mesenchymal stem cellsinto a strip with two ends and transfecting the mesenchymal stems cellswith a nucleic acid encoding as a sodium channel so that the mesenchymalstem cells functionally express the sodium channel, wherein the stripcan be attached to a heart so as to create a tract between an atrium ofthe heart and a ventricle of the heart, which tract is capable ofpropagating electrical signals.